Staphylococcus aureus (MRSA) has increased sharply in Hospital District of Southwest Finland (HD). To understand reasons behind this, a retrospective, population-based study covering 10 years was conducted. All new 983 MRSA cases in HD from January 2007 to December 2016 were analysed. Several data sources were used to gather background information on the cases. MRSA cases were classified as healthcare-associated (HA-MRSA), community-associated (CA-MRSA), and livestock contact was determined . Spa typing was performed to all available strains. The incidence of MRSA doubled from 12.4 to 24.9 cases/100000 persons/year. The proportion of clinical infections increased from 25 to 32% in the 5-year periods, respectively, (p < 0.05). The median age decreased from 61 years in 2007 to 30 years in 2016. HA-MRSA accounted for 68% of all cases, of which 32% associated with 26 healthcare outbreaks. The proportion of CA-MRSA cases increased from 13% in 2007 to 43% in 2016. Of CA-MRSA cases, 43% were among family clusters, 32% in immigrants and 4% were LA-MRSA. The Gini-Simpson diversity index for spa types increased from 0.86 to 0.95 from the first to the second 5-year period. The proportion of a predominant strain t172 decreased from 43% in 2009 to 7% in 2016. The rise in the proportion of CA-MRSA, the switch to younger age groups, the complexity of possible transmission routes and the growing spa-type diversity characterize our current MRSA landscape. This creates challenges for targeted infection control measures, demanding further studies.The incidence of methicillin-resistant  Staphylococcus aureus (MRSA) infection remain a global concern [High and increasing rates of methicillin-resistant  concern , 2. Neve concern . We condThe hospital district of Southwest Finland (HD) is a public joint municipal healthcare authority, consisting of 28 member municipalities with a total of 478,500 residents. The population of HD represents 8.7% of the population in Finland (5.5 million).All clinical microbiology laboratories in Finland notify new MRSA findings from both clinical and screening samples to the National Infectious Disease Register (NIDR) maintained by the Finnish institute for health and welfare (THL). The Turku University Hospital clinical microbiology laboratory notifies MRSA cases also to a regional Register of Hospital Infections and Carriers of Resistant Bacteria  kept by HD. An individual with a positive MRSA finding (MRSA case) is notified to SAI only once irrespective of the number of positive cultures after the first notification. In each new MRSA case, hospital contacts and risk factors for transmission are tracked by an infection control nurse. The patient records of hospital contacts are tagged with screening alert for future admissions.All MRSA cases in HD from January 2007 through December 2016 were included in the analysis. Predisposing factors for MRSA carriage were obtained from the SAI register, patient records and a patient questionnaire designed for identifying community-associated MRSA . The incMRSA cases in healthcare workers (HCWs), infants under 28 days of age, and patients who were hospitalized or who had stayed in a nursing home within the previous 2 years in Finland, or 1 year abroad, were defined as healthcare-associated MRSA (HA-MRSA). Cases without such criteria were defined as community-associated MRSA (CA-MRSA) if MRSA was isolated at outpatient care or within the first 2 days of hospital care. If MRSA was isolated later during hospital care, the case was defined as HA-MRSA. Clinical samples were defined as diagnostic bacterial culture from the site of infection whereas screening samples were taken from asymptomatic individuals according to screening protocols. Each case was defined either “clinical infection” or “asymptomatic carrier” based on the sample type from which MRSA was first identified.Healthcare outbreaks were defined as two or more MRSA cases in patients who had shared a room or washing facility in a healthcare ward and MRSA cases in HCWs working in the same ward. Family clusters were defined as two or more MRSA cases living in the same household. For each outbreak and cluster, the index case was defined as the first identified case leading to outbreak or cluster investigation. Livestock-associated MRSA (LA-MRSA) cases were defined as MRSA cases in pig farmers, their household members or employees working at the farm.In addition to standard precautions, infection control (IC) practices in HD to prevent MRSA outbreaks include targeted screening of patients attending hospital care, barrier precautions and isolation rooms in the care of MRSA carriers, as well as MRSA cohort units in long-term care. At admission to hospital, screening samples are routinely taken from patients who have stayed in a hospital or nursing home abroad within previous year prior to hospitalisation, and from asylum seekers (targeted screening since 2015). When a new MRSA case has been identified in hospital or long-term care, all exposed patients, determined by a shared room or toilet or washing facility within the previous year, are traced and screened promptly or at next hospital admission. In Finland, screening is also performed in patients exposed in a hospital or nursing home with an MRSA outbreak. HCWs are screened in case of an unresolved MRSA outbreak in a ward or after working in healthcare facilities abroad within the previous year. Household contacts of MRSA carriers are screened before decolonisation of the carrier and at admission to hospital. MRSA cultures for screening are taken from the anterior nares, throat, armpits, groins, perineum, and skin lesions.S. aureus identified according to standard methods, including rapid coagulase tests, Vitek2 and since 2011, Maldi-TOF MS. Methods for MRSA surveillance varied during the study period but included use of chromogenic MRSA-agars (since 2007) and enrichment broth (since 2008). Cefoxitin was used for selection of MRSA in both enrichment and antimicrobial susceptibility testing by disk diffusion according to CLSI and since 2011 the EUCAST methodology. The presence of mecA  was confirmed by a molecular method throughout the study period.Clinical samples were inoculated, and spa typing since 2009 to date. Spa typing was performed as described elsewhere [spa sequence was analysed by using the Ridom StaphType™ software .Finnish clinical microbiology laboratories send all new MRSA isolates to THL for strain typing. The standard methods for MRSA typing were pulsed field gel electrophoresis (PFGE) in 2007–2008, and lsewhere , 6 for aspa types was estimated for time periods 2007–2011 and 2012–2016 (the former and the latter 5-year period). In addition to the overall diversity, diversity was calculated separately for HA-MRSA and CA-MRSA cases, and similarly for clinical and screening samples. Demographic variables of the MRSA carrier population were compared between the two time periods using chi-squared test or Fisher’s exact test. For each spa type carried by at least 10 persons, its association with the HA-MRSA/CA-MRSA and clinical infection/asymptomatic carrier case status was evaluated using chi-squared test or Fisher’s exact test. Data analyses were performed with IBM SPSS Version 25 for Windows  and with R for Windows, version 3.6.0.Trends in median age and in the proportion of HA-MRSA/CA-MRSA cases were studied using median regression and the chi-squared test for trend in proportions, respectively. The Gini-Simpson diversity index of The median age of MRSA cases was 43 years range 0–103 years). The median age decreased from 61 years in 2007 to 30 years in 2016, corresponding to a 2.6-year decline in the median age per calendar year (95% CI 1.5–3.3). The proportion of cases ≥ 65 years of age decreased (range 20–47%/year) but the proportion of cases under 15 years of age remained relatively low (range 6–18%/year) throughout the study period . The incidence of MRSA increased 2.0-fold from 12.4 to 24.9 cases/100000 persons/year during the study period. In the whole country, the incidence of MRSA increased 1.3-fold from 24.1 to 31.2 cases/100000 persons/year during the same time period   were diagnosed from clinical samples taken from the site of infection. The proportion of clinical infections was higher in the latter 5-year period as compared with the first 5-year period  MRSA cases were classified as HA-MRSA . Five of the healthcare outbreaks occurred in the University Hospital, seven in municipal health centre wards and 14 in long-term care facilities. There were thirteen outbreaks in both 2007–2011 and 2012–2016, although there was a decrease in the number of cases linked to the outbreaks .Of all HA-MRSA cases, long-term care facility residents accounted for 26% (176/673) and infants under 28 days of age for 1% (9/673). The remaining 63% (419/673) had been in hospital care, of which 32% (134/419) abroad. One-third  had an epidemiological link to an outbreak in a healthcare facilityHCWs accounted for 10% (69/673) of the HA-MRSA cases, and their proportion of all MRSA cases decreased from 9% (40/445) in the first 5-year period to 5.4% (29/538) in the latter  MRSA cases were classified as CA-MRSA. The proportion of CA-MRSA of all MRSA cases was 13% (8/62) in 2007 and 43% (56/130) in 2016, corresponding to a 7.5% increase per calendar year (95% CI 2.6–12.7) in the odds of CA-MRSA . Of the 121 index cases, 70% (85/121) were HA-MRSA and 30% (36/121) CA-MRSA. Of the secondary household cases, 37% (67/183) were HA-MRSA and 63% (116/183) CA-MRSA. Table We identified a total of 213  MRSA cases among the non-native/foreign-born population in HD, including asylum seekers and refugees, other immigrants and residents of foreign countries. Of those, 15% (31/213) were detected by clinical samples. Refugees accounted for 86 cases, of which 11 cases were detected during 2007–2011, and 76 cases in 2012–2016. Other immigrants accounted for 11% (113/983) of the cases, of which 25 and 88 cases were identified in the first and in the second half of the study period, respectively. MRSA was identified in six residents of foreign countries and in eight foreign healthcare exchange students.Altogether 22  livestock-associated MRSA (LA-MRSA) cases were detected, the first ones in 2010. MRSA findings were detected from screening samples in 77% (17/22) of the cases, and from clinical infection sites in 23% (5/22) of the cases. Nine cases had also a healthcare association.We identified 11 cases  with a documented history of intravenous drug abuse either previously or presently. Their proportions of all cases in the two 5-year periods were 0.9% and 1.3%, respectively (Table Spa typing was performed on 99.3% (976/983) of the MRSA strains. Seven strains (0.7%) from years 2007 and 2008 were not available for typing. Altogether 173 different spa types were identified while 12  strains remained non-typeable (NT). Fifteen spa types were identified from ≥ 10 cases, representing 70% of the strains, 60 spa types from 2 to 9 cases (19%), and 98 spa types were sporadic (10%). The most common spa types were t172 , t008  and t002 . Spa type t172 was predominant throughout the study period, but its proportion first increased from 18% in 2007 to 43% in 2009, then decreased steadily down to 7% in 2016. The Gini-Simpson diversity index increased from 0.86 (95% CI 0.82–0.88) to 0.95 (95% CI 0.94–0.96) from the first to the second half of the study period , as well as between clinical infections and asymptomatic carriage groups . Spa types t032, t040, t688 and t067 were associated with HA-MRSA (p < 0.005), whereas spa types t002, t019, t034, t044 and t223 associated with CA-MRSA (p < 0.04). Spa types associated with clinical infections were t032 (p < 0.001), t044 and t127 (p < 0.05). Spa type t172 predominated in both HA-MRSA  and CA-MRSA  groups, as well as in healthcare outbreaks  and in family clusters , but not in the immigrant group , where the most common spa types were t223 and t044 . The LA-MRSA spa types were t034 , t2741 , t108 , t1250 and t032 .The spa types between the different MRSA groups. This complicates planning of preventive actions against spreading of MRSA, especially in the community setting.In this study, we document an increase in the proportion of CA-MRSA in our hospital district (HD). Simultaneously, the boundaries between epidemiologically defined CA- or HA-MRSA cases have become increasingly blurry, indicated by the observed overlap of The incidence of MRSA doubled from 2007 to 2016 in our HD, still remaining below the national average level. Most of the increase is due to CA-MRSA. Previously, the incidence of CA-MRSA isolations had tripled in Finland in 1997–2006, but proportion of CA-MRSA decreased as the total amount of MRSA cases eightfolded , 7. Reflspa-type diversity increased significantly in addition to the substantial decline of a previously dominant strain type t172. Previously, t172 was associated to CA-MRSA [spa types have been reported in other countries of low MRSA incidence [spa types, the increased diversity is, at least partly, indicative of human migration and travelling [The  CA-MRSA , but in ncidence , 10, 11.avelling , 13. Theavelling , 15.S. aureus findings [Clinical infections accounted for only 28% of all cases in our study. However, the proportion was on the rise in the latter period of the study. The prominence of asymptomatic carriers is common where an active “search-and-destroy” screening policy is exploited . The scrfindings .There was a declining trend in the proportion of HA-MRSA and the outbreaks in healthcare facilities were smaller in the latter period of the study. In 2007, 87% of MRSA cases were associated with healthcare, whereas in 2016 the proportion was only 57%. After large MRSA outbreaks in hospitals and long-term care facilities in the 1990’s , the freMRSA findings in HCWs differed significantly between the two study periods. In the first period, HCWs were screened in uncontained hospital outbreaks, but in the latter period, in absence of hospital outbreaks, the need for screening diminished and HCWs were mainly screened only when having a history of work at hospitals abroad.Intravenous drug users were not routinely screened for MRSA, and only those with a well-documented history of drug abuse in patient records were regarded as intravenous drug users. Thus, our number of MRSA findings in intravenous drug users is not conclusive.A prominent MRSA group appeared to be household members of MRSA carriers. Our screening practice including household members of MRSA carriers proved very effective as altogether 19% (183/983) of MRSA cases were detected that way. A high risk of MRSA transmission between household members has been noted earlier , 25. In spa types, and furthermore, whole genome sequencing (WGS) of the family cluster-associated isolates could shed light on transmission routes.Interestingly, a significant proportion of MRSA-positive household members of an index case in family clusters also possessed a contact to healthcare . A detailed analysis on the spa-type diversity most likely reflect similar movements in the population.The sharp increase in the numbers of refugees and asylum seekers in Europe in 2015 affected also Finland. Compared with 2014, the number of refugees ninefolded in Finland in 2015 yet declining sharply thereafter. The proportion of population from foreign origin in our HD was 4% in 2007 and 7% in 2016 . Previouspa types among the LA-MRSA cases, 21/22 (95%) have previously been linked to livestock. Spa types t034, t108 and t1250 have been linked to livestock in Europe [Since 2010, pig farmers and their families along with farm workers emerged as a distinct but a relatively small group within our MRSA carrier population. Cases associated with pig farming (LA-MRSA) were mostly found in the last 2 years of the study. In comparison with Denmark and the Netherlands in Finland, the human LA-MRSA situation remained still moderate during our study years , 33. Thin Europe , 36. Typspa-type distribution between the HA- and CA-MRSA groups, those differences could have multiple explanations, strains originating from abroad being the most likely cause. Moreover, the most prevalent spa type t172 in our HD has previously been associated with CA-MRSA [Our study shows that the often-used epidemiological classification into HA-MRSA, CA-MRSA and LA- MRSA is too rigid as MRSA transmission may occur through multiple and interplaying sources. For example, a part of HA-MRSA cases in our study may in fact have originated from the community because, by definition, we allowed a rather long period (up to 2 years) between recorded hospital care period and the subsequent MRSA finding. Furthermore, although we did find some differences in the  CA-MRSA , but thi CA-MRSA .spa typing of MRSA strains for a 10-year period. Detailed review of data files of each MRSA case could be performed which improves the quality of analysis. Restricting the study to one hospital district alone means that the similar practices over the long study period could be employed, and any changes were traceable.The strength of this study was the use of multiple data sources, including epidemiological data, patient interviews, patient records and universal In conclusion, HA-, CA- and LA-MRSA strains intermingle in community and healthcare, making analyses of exact transmission routes difficult. The prevention of the spread of MRSA may comprise diverse measures in community and in healthcare. Effective means of preventing MRSA in community include strict antibiotic policy , 39 and


The current study aimed to assess the amount of fluoride released from fluoride-containing dental adhesives and its effect on micro-tensile bond strength (µTBS) and on resistance to demineralisation of dentine and enamel.Two fluoride-containing dental adhesives, and a fluoride-free adhesive were used as experimental adhesives. After thermal cycling the µ-TBS of adhesives to dentine and the failure mode were assessed. The fluoride release and cross-sectional microhardness (CSMH) of specimens were measured before and after one day, 7 and 28 days of pH-cycling. The data were analysed using one-way ANOVA, Weibull statistics and repeated measures ANOVA. p≤0.001). The CSMH values of both the dentine and enamel underneath the adhesives was reduced at 28 th day of the pH-cycling compared to the baseline (p≤0.001). From day 1 to day 28, the released fluoride declined in both the fluoride containing dental adhesives (p≤0.001).The results indicated a significant difference between the group of FL and both the SE and LBF groups (Based on the results, the released fluoride from dental adhesives may adversely influence the bond strength and durability of the resin/dentine interface. Moreover, the released fluoride didn’t improve the resistance to demineralisation of adjacent enamel and dentine to bond interface. Key words:Fluoride release, micro-tensile bond strength, microhardness, fluoride-containing adhesives. The teeth then were randomly divided to different groups for different test methods.- Microtensile bond strength testThe selected teeth were divided to three groups (n=5) according the adhesive system used. The occlusal surfaces of teeth and the teeth roots were removed using a diamond saw . The surfaces of exposed dentine were observed with a stereomicroscope  for evaluation of any pulp horns and for inspection of any remaining enamel. Afterwards using 600 grit silicon carbide paper a standardized smear layer was created on the prepared dentine surfaces.The adhesive systems including a fluoride free self-etch adhesive Clearfil SE Bond  and two fluoride-containing self-etch adhesive, Fluorobond II  and Clearfil Liner Bond F, were applied on the exposed dentine surfaces according to the manufacturer’s instructions. A 6-mm-thick build-up of composite resin  was incrementally (in 2-mm-thick layers) light polymerized using an LED light-curing unit  with an output of over 1200 mW/cm2 for 20 s. The manufacturers, compositions, lot numbers and manufacturer’s instructions of the materials used in this study are listed in The prepared samples were then stored in artificial saliva at 37°C for 24 hours. Thereafter the teeth were sectioned serially parallel to long axis of teeth in mesio-distal and bucco-lingual directions at 1-mm intervals using a water-cooled diamond saw . At least six 1 × 1× 12-mm2 longitudinal rectangular sticks with two halves of resin composite and dentine were obtained from each tooth. The beams were then subjected to 3000 thermal cycles between 5°C and 55°C with a dwell time of 20 s.The beams then were fixed in a microtensile testing apparatus with a cyanoacrylate adhesive and subjected to microtensile testing using a universal testing machine with a 6.0-kg load cell  at a crosshead speed of 1mm/min. ‘Pre-testing failures’, including debonded samples during the thermal cycling were not considered in the statistical analysis .The fractured sticks of debonded specimens were gold coated and observed under scanning electronmicroscopy  to determine the mode of failure. Failure modes were classified into four groups described below.1) Adhesive at the dentine-resin composite interface 2) cohesive failure in dentine 3) cohesive failure in resin composite 4) mixed failure.- Cross-sectional microhardness testFor a microhardness test, 39 healthy non carious teeth were randomly divided into three groups according to adhesive system.2 mm depth, 3 mm occlusogingival high and 4 mm mesiodistal width Class V cavities were prepared using a flat-ended cylindrical bur and a high speed equipment under water cooling on the buccal or lingual surfaces of the human third molars. The occlusal and cervical margins were located in enamel and dentine respectively. After preparation of five teeth the bur was replaced with a new one to maintain the uniformity of cavities.The adhesive systems were applied according to the manufacturer’s instructions on the surfaces of prepared cavities. The composite resin  was then incrementally placed (1-mm-thick layers) to the cavities and light polymerized using an LED light-curing unit  for 20s.Afterwards the teeth with their restored cavities were sectioned mesiodistally into four slabs using a diamond saw . The slabs were assigned into the four groups according to the days of pH-cycling process: 0 day, 1 day, 7 days and 28 days. In vitro pH-cycling scheme was used for 156 obtained slabs including 18 h in remineralisation solution and 6 h in demineralisation solution on each day . The demThe enamel slabs then were embedded in polymethyl methacrylate, and hand-polished using silicon carbide papers up to 2,000 grit to produce a flat surface. A cross-sectional microhardness (CSMH) using a microhardness tester  with a Vickers diamond indenter was carried out to evaluate the demineralisation of enamel and dentine adjacent to adhesive resin. The microhardness measurement of enamel slabs was done prior to the pH-cycling procedure, one day after pH-cycling, seven days after pH-cycling and 28 days after pH-cycling. Six Vickers Pyramid Numbers (HV) from each sample were recorded under a load of 200 g and 50 gr for 15 s in enamel and dentine respectively.- Fluoride releaseThe De/Re solutions of individually pH-cycled specimens of Clearfil Liner Bond F  and Fluorobond II  adhesive systems after a day, 7 days and 28 days of pH-cycling procedure were collected for fluoride release measurement.The fluoride concentration of solutions was determined using the ion-selective electrode (ISE) method. A calibration procedure first was performed. A 100 p.p.m. standard solution of sodium fluoride  with deionized water was prepared and stored in a clean plastic container. A series of 12 x dilutions including solutions of 100, 50, 25,10, 5, 2.5, 1, 0.5, 0.05, and 0.01 then were produced and the calibration graph plotted  (Merck company) was added to 500 ml distilled water and 40 % sodium hydroxide also was added drop by drop to dissolve the salt. Then 300 g sodium citrate dehydrate and 60 g sodium chloride were mixed with the solution under stirring. The final volume of solution was adjusted to one liter by adding distilled water.The fluoride concentration of solutions was measured using a pH/Ion meter  attached to an ion selective electrode for fluoride ) and a graph of the meter reading vs. known concentration of fluoride was plotted.- Statistical analysisAfter confirmation of normal distribution of data, the one-way analysis of variance (ANOVA) followed by a post-hoc Tukey’s test was performed to determine the micotensile bond strength of three different adhesive systems. A repeated measures ANOVA followed by a Bonferroni post hoc test was used to determine the CSMH and fluoride release of samples before pH-cycling and a day, 7 days and 28 days of pH-cycling process. All statistical analyses were performed at significance level of 0.05 using SPSS 23.0 for Windows.The bond strength data were also statistically analysed using Weibull analysis . The Weibull modulus and also characteristic strength (63.2% unreliability) were calculated by the estimation of Rank Regression on X (RRX) and Fisher Matrix Confidence Bounds (FM). The 10% and 90% unreliability levels of the specimens of three adhesives also compared by ordering the bond strength values from the lowest to the highest value and using the following equation: Pf=(i-0.5)/nwhere i is the i th datum and n is the total number of data points of each group- Microtensile bond strengthp=0.28). However, the results indicated statistically significant difference between the group of FL and both SE and LBF groups (p≤0.001).The mean microtensile bond strength values, standard deviation, and Weibull parameters of a fluoride-free and two fluoride-containing self-etch adhesives are presented in The obtained characteristic strength of groups ranged from 35.92 for the FL fluoride containing group to 56.49 for LBF fluoride containing dental adhesive, as shown in Failure analysis using SEM revealed that the fracture pattern in most of the specimens of SE bond was cohesive into the bulk of resin composite (60%) Fig. . On the - Cross-sectional microhardness (CSMH)The mean hardness values and standard deviations of experimental groups of dentin and enamel sections at baseline and after pH-cycling regime are described in The mean CSMH values and standard deviations of samples before pH-cycling procedure and after seven days and 28 days of pH-cycling at dentine and enamel are described in p≤0.001). On the first day of pH-cycling, the FL bond group also showed trend similar to SE bond group; and the mean hardness value significantly reduced after a day of pH-cycling (p≤0.001), however, this reduction was not statistically significant between the first and seventh days of pH-cycling (p=0.684). The CSMH values in FL bond samples increased after day 7 up to day 28 as SE bond group, although this difference was not statistically significant (p=0.169). The changes in hardness values of dentine in the LBF group showed a behaviour similar to that of the SE bond group up to the day 7 (p≤0.001). However, unlike the SE bond and LBF groups, the hardness value of dentin slightly decreased up to day 28 (p=0.759).As shown, the time of pH-cycling significantly affected the hardness values of the dentine underneath the SE bond adhesive. In this regard, the CSMH values declined up to day 7. After that, the hardness values significantly increased at day 28 of the pH-cycling process (p<0.05). However, a slight increase was observed between day 1 and day 7 in the FL bond and LBF groups (p=1.00). The profiles of changes in microhardness of enamel and dentine underneath the adhesive systems during the pH-cycling regime are shown in Fig. The time of pH-cycling process also had a significant effect on the enamel hardness underneath the self-etch adhesive systems. Accordingly, the enamel hardness values of all experimental groups decreased until day 28 of pH-cycling p<0.0. However- Fluoride release:p≤0.001). LBF adhesive also showed a trend similar to that of the FL bond; the released fluoride reduced from day 1 until day 28 (p≤0.001). However, the slope of the profile was blunter than the FL bond.The profiles of fluoride release from each fluoride-containing dental adhesive at different experimental times of pH-cycling procedure are shown in Fig. Many studies have shown the caries prevention effect of fluoride ,17. Due Accordingly, the aim of this study was to assess the fluoride release and dentine bond strength of self-etch fluoridated dental adhesives, and also the resistance to demineralisation of the dentine and enamel adjacent to these adhesive systems.et al. (et al., (et al. (et al. (et al. (et al. (et al. (The results of the study showed significantly higher dentine bond strength in the LBF and SE groups compared to the FL group. A meta-analysis review of the performance of the ten most common dental adhesives presented the Clearfil SE Bond as the second best-performing adhesive . Clearfiet al.  reportedet al. . Lida et(et al.,  also rep(et al., . The hig (et al. , found a (et al. . Nakajim (et al.  attribut (et al. . Despite (et al. , who rep (et al. . As we o (et al. ,28. Neve (et al.  also rep (et al. . Similar (et al. , who did (et al. . Accordi (et al. ,32. The Within the limitations of the current study, it can be concluded that fluoride released from fluoride-containing dental adhesives did not affect the resistance to demineralisation of human tooth structure. Moreover, the released fluoride may adversely influence the integrity of resin/dentine interface and, hence, the bond strength and bond durability of adhesive system to dentine or enamel structure.


Plasmodium falciparum. Significant human RBC engraftment was achieved by ameliorating the residual non-adaptive immune response using clodronate-loaded liposome treatment. Controlled reactive professional phagocytic leukocytes in immunodeficient mice allowed for sizeable human blood chimerism and injected huRBCs acted as bona fide host cells for P. falciparum. huRBC-reconstituted immunodeficient mice received infectious challenge with attenuated P. falciparum C9 parasite mutants (C9-M), complemented (C9-C), and wild type (NF54) progenitors to study the role of immune effectors in the clearance of the parasite from mouse circulation. C9-M and NF54 parasites grew and developed in the huRBC-reconstituted humanized NSG mice. Further, the presence of mutant parasites in deep-seated tissues suggests the escape of parasites from the host's immune responses and thus extended the survival of the parasite. Our results suggest an evasion mechanism that may have been employed by the parasite to survive the mouse's residual non-adaptive immune responses. Collectively, our data suggest that huRBCs reconstituted NSG mice infected with attenuated P. falciparum is a valuable tool to explore the role of C9 mutation in the growth and survival of parasite mutants and their response to the host's immune responses. This mouse might help in identifying novel chemotherapeutic targets to develop new anti-malarial drugs.A recently developed humanized mouse has been used to assess the immune response evoked against the isolated attenuated C9 parasite clone  of PF3D7_1305500) of  However, the study of human malaria parasites in animal models is severely limited by ethical and technical constraints, since only a few primate species have been found to be receptive to P. falciparum infection  which are used as surrogates to study human malaria  parasites grafted in humanized mice showed nearly similar parasitemia patterns to that of NF54. Further, the C9-M parasite seems to have employed a mechanism to evade the host's immune responses and resides/sequesters in the deep-seated tissues. Our study showed the importance of human RBC reconstituted NSG mouse model to study the behavior of generated attenuated and complement parasites  mice were procured from Jackson Laboratory, USA. The transgenic thymidine kinase-NOG (TK/NOG) strain was procured from Taconic, USA. The immunodeficient/transgenic mice were housed in sterile isolators and supplied with autoclaved tap water with a γ-irradiated pelleted diet ad libitum. Animals were manipulated under pathogen free conditions using a laminar flow-hood cabinet. One hundred and ten animals were used in the study. Six animals were allocated for each experimental group, except for the giemsa staining assay wherein four animals were used. Animal allocation was randomized, and mice receiving treatment were monitored daily and weighed three times  per week. P. falciparum infected mice were treated with the analgesic ketoprofen to alleviate pain caused by the malaria infection.Four- to 6-week-old male and female NOD/SCIDIL-2Rγbona fide host cells for the development of P. falciparum in in vitro cultivation and huRBCs reconstituted mice. The packed huRBCs were provided by the Interstate Blood Bank (Chinese blood bank). Blood was taken from donors with no history of malaria. The huRBCs were suspended in SAGM  and stored at 4°C for a maximum of 2 weeks. Before injection, huRBCs were washed three times in RPMI-1640 medium , supplemented with 1 mg of hypoxanthine per liter , and warmed for 10 min at 37°C. Blood samples drawn from mice were used to determine the percentage of huRBC in mice's peripheral blood at regular intervals by flow cytometer (Accuri cytometers) using FITC labeled anti-human glycophorin antibody .The huRBCs are the P. falciparum line (NF54) were employed in the study. C9-M and C9-C parasites were generated by the interstional mutagenesis by others , 35 mM HEPES (Sigma), 0.25% NaHCO3, 0.5% albumax II (Gibco/BRL), and 0.01 mg/ml gentamicin.C9 mutant, complement parasites (Gene complementation of the C9 mutant to rescue the wild-type phenotype) and wild type y others . P. falclsewhere . The parP. falciparum was maintained in huRBCs reconstituted NSG (immunocompromised) mice undergoing additional modulation of innate immune-defenses using the clodronate-containing liposomes, as described earlier  of mouse erythrocytes (45fL), MCV of human erythrocytes (86fL), hematocrit in the mice of 0.7, weight of NSG mice (25 grams), and a conservative estimate of 5.5 ml of blood per 100 grams of mouse weight using the following equation:Number of infected RBCs = (0.055 ml/g) (25 g) (0.7) × (huRBC parasitemia)[86fL + (mouse Chimerism/human Chimerism) 45fL]Parasite cultures at 5% parasitemia, predominantly ring stages, were synchronized to remove late stage parasites using pre-warmed (37°C) 5% sorbitol. After harvesting culture at 1600 RPM for 6 min, medium was removed and replaced with 10 volumes of pre-warmed 5% sorbitol . The re-P. falciparum infected mice were euthanized to extract organs . These extracted organs were perfused, and cells collected from the organs were placed on the glass slides. Briefly, cells were fixed with methanol and stained with Giemsa, and examined at 100X magnification to perform differential counts of each stage (200 parasites from each organ counted). The analysis of deep-seated organs for parasite count in NSG mice infected with C9-M and C9-C parasites was carried out.in vitro cultures as well as from humanized mouse (huRBCNSG-IV) infected with C9-M, C9-C, and wild type parasites. The specific primers  and  were used to identify the presence of hDHFR and BSD from in vitro cultures and P. falciparum-harboring humanized mice. The genomic DNA was extracted from parasite cultures and tail snips of mice infected with P. falciparum strains using a DNA mini-kit (Qiagen). DNA was amplified in 20 μl reaction mixture by adopting the following PCR conditions: 1 cycle at 45°C for 30 min and 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 45°C for 30 s, and 65°C for 3 min. The amplified DNA showed the product band of 563 and 393 bp on 0.8% agarose gel of hDHFR and BSD, respectively.The original parasites C9-M and C9-C were harvested from P. falciparum wild-type and mutant parasites at 0.5–2% parasitemia in 96-well plates and diluting every 48 h for 168 h. The parasite cultures were plated in triplicate at time zero and end point of the assay, and for each time point samples were taken at every 24 h for 7 days and fixed with 0.05% glutaraldehyde after the removal of culture medium. Flow cytometer was used to estimate the parasitemia as described elsewhere  following the manufacturer's recommendations.Hundred microliter blood samples were collected through the submandibular puncture of mice, and sera were stored at−80°C. Cytokines and chemokines  were quantified using the BDTransgenic offspring were genotyped and identified by PCR  using HTKF1 forward primer, 5′-CACGTCTTTATCCTGGATTACG-3′ and hGHR1 reverse primer, 5′-CACTGGAGTGGCAACTTCCA-3′. The genomic DNA was extracted from tail snips by DNA mini-kit (Qiagen), and amplified in a 20 μl reaction mixture using the PCR conditions: 2 min at 94°C, then 30 cycles of 30 s at 94°C, 30 s at 59°C, and 30 s at 72°C, and finally 3 min at 72°C. The transgene DNA showed an amplified product band of 236 bp on 1% agarose gel .t-test using Prism software (Graph Pad 5 Demo) and data was expressed as the mean ± standard deviation (S.D.) of the mean . A value of P < 0.05 was considered statistically significant. The evaluation of inflammatory mediators' results are presented as mean SEM from the experiment performed using three mice per group.Each growth assay data was analyzed using C Flow Plus software (Accuri). Growth rate and statistical analysis was carried out by Student's P. falciparum growth. Experiments were performed in NSG mice using 650 μl of huRBC pellets mixed with 25% de-complemented human serum intravenously injected three times every week, and reconstituted NSG mice were infected with P. falciparum through an intravenous route. Codronate-loaded liposome (clo-lip) suspension was injected intraperitoneally, and clo-lip and huRBCs were administered the same day. This mouse model is called “Pf-NSG IV” model. With this IV protocol 100% of NSG mice, as reported earlier  numbers of huRBCs in mouse periphery supported rapid and optimum growth of P. falciparum  differences in parasite growth in NF54, C9-M, and C9-C parasites in these humanized NSG mice. The short-term human blood chimerism in mouse circulation led to the clearance of NF54 infected erythrocytes on day 13 and 14 post-infection   using random insertional mutagenesis with a piggyBac transposon (in vitro culture of intra-erythrocytic C9-M mutant parasites was seen and compared with the wild type (NF54) parasites  , we nextansposon . Fifty parasites . The muth others , the meah others  parasiteh others . Growth h others . The groin vitro  parasite when growth assays were conducted on parasites from culture and grafted humanized mice. Therefore, we decided to characterize both mutant and complement parasite strains. Parasite strains grafted in humanized mice (Pf-huRBC/NSG-IV) were typed for the attenuated C9-M and C9-C phenotype by PCR . Both stin vitro , and theNSG mice . hDHFR (NSG mice  and BSD NSG mice  showed ap < 0.05) human blood chimerism (proportion of huRBC in mouse's periphery) ranging from 60 to 70% of total erythrocytes. This blood-chimerism was stable over a month and supported rapid and optimal growth of P. falciparum  in huRBCs reconstituted humanized mice. Following the confirmation of the development and replication of typed mutant (PF3D7_1305500) parasite in humanized mice, upon injection with 650 μl of huRBC, our results showed the significant . Interestingly, a greater increase in TNF-α  and IL-12  was seen in the mice infected with C9-C parasites than those seen with NF54 infected mice  of huRBC showed an initial establishment of human blood chimerism followed by a decrease, which is most likely due to the inflammation induced by parasites . Therefolciparum . The higted mice . The subted mice , the iniCells of monocyte-macrophage lineage are continuously recruited and play an important role in the clearance of the parasite from mouse circulation, but mutant parasites reside for long periods of time. Therefore, we next decided to confirm the importance of monocyte/macrophage in the clearance of parasites. The macrophages are the main subsets recruited all over the course of infection, and found to be more active than other phagocytes at ingesting infected and uninfected huRBCs in the peritoneum , 34–36 . C9-M pain vitro culture, which was supported by the humanized mice to induce the production of gametocytes  strain of lciparum . All micP. falciparum strains tested  to study unknown asexual blood stage genes/RNA binding proteins of h others , our in  studies . Moreovezed mice .P. falciparum strains in a PfhuRBC-NSG-IV humanized mouse  may be useful to study the immune responses evoked against the grafted parasite, and possible survival mechanisms employed by the parasites. The additional defects in the innate immune system of NSG mice are most likely related to the defective activation of phagocytes which led to the reduced P. falciparum selection pressure  evolution of parasitemia in mouse's periphery remained stable but short-term blood chimerization (insufficient huRBCs) in the circulation leads to the clearance of parasites driven by the pro-inflammatory cytokines; (3) growth and development of all P. falciparum strains was supported by the developed humanized mice, indicating that, in contrast to Aotus, Saimiri, and previous mouse models, there is no requirement for the preliminary adaptation of the parasite to PfhuRBC-NSG-IV host. The slower growth of C9-M indicated that the parasite could retain the mutant phenotype upon being grafted in human RBCs reconstituted humanized mice. However, there was no difference seen in the parasitemia patterns of grafted parental NF54, C9-M, and C9-C parasites. This shows the relevance of this mouse as it helps in understanding the behavior and relevance of C9 mutation in the growth and replication of the asexual blood stage infection of P. falciparum without tampering its genotype and phenotype; (4) migration of parasites from the mouse's periphery to deep-seated organs with the extended residence indicated that C9-M parasites may have employed molecular mechanism(s) to partially evade the host's innate immune response; (5) humanized mouse supported the development of the sexual stage of P. falciparum. Stage III, IV, and V gametocytes were seen in the peripheral blood but their infectivity could not be demonstrated; and (6) transgenic/immunodeficient (TK/NOG) mice were shown to better control the non-adaptive immune response as compared to NSG mice, and therefore higher human blood chimerism was seen when reconstituted with huRBCs in the mouse prepared by clo-lip treatment. huRBCs grafted TK/NOG mice showed greater susceptibility toward infection with stable parasitemia in all P. falciparum strains tested (Our results show that: (1) s tested .Results obtained with the humanized mouse model, as reported earlier , 13, 16,P. falciparum. The in vitro findings  and immune-suppressor (IL-10) than their wild type progenitors was seen. We believe that improved survival of infected and uninfected huRBCs is associated with a decrease in the inflammatory cytokine and a slight increase in the production of IL-10 by the cells of myeloid origin in C9-M harboring mice. Therefore, controlled pro-inflammatory cytokines allowing the sustained parasite development in deep-seated organs indicate that immunity . We measimmunity . The incimmunity , 56, 57,rophages , 58 alsolciparum .P. falciparum-NSG-IV model may allow the study of asexual blood stage growth mutant(s) and their effect on the human system. This mouse shows reproducibility of both huRBC grafting and parasite survival, less day to day variation in parasitemia, does not require preliminary adaptation of parasite strains to the mouse, and supports the development of even attenuated P. falciparum. The evasion mechanisms employed by the parasites help survive against the host's residual immune responses and raises the possibility of partial sequestration of parasites in deep-seated organs, which are key findings of the present work. The delineation of in vivo function and behavior of attenuated C9-M parasites gene (PF3D7_130550) in a humanized mouse might give insights into this unknown protein, important for the growth and development of the asexual blood stage of P. falciparum.The emergence of resistance to frontline drugs and the lack of diverse therapeutic agents necessitates the finding of effective anti-malarial drugs and the identification of new drug targets , 59–65. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The Second Hospital of Jilin University, Changchun, China.LZ and MT conceived and designed the experiments, analyzed the data, and wrote the paper. LZ, J-LL, M-XJ, DT, L-YW, and CC performed the experiments and contributed materials/analysis tools. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The materials were found to be characterized by very high antibacterial activity against S. aureus and K. pneumoniae. Cytotoxicity studies confirmed that the materials are not cytotoxic. Copper alginate conjugates with RGD peptides have strong potential for use in regenerative medicine, due to their biocompatibility and innate antibacterial activity.Current restrictions on the use of antibiotics, associated with increases in bacterial resistance, require new solutions, including materials with antibacterial properties. In this study, copper alginate fibers obtained using the classic wet method were used to make nonwovens which were modified with arginine-glycine-aspartic acid (RGD) derivatives. Stable polysaccharide-peptide conjugates formed by coupling with 4--4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO Alginate is a naturally occurring polysaccharide composed of 1→4) linked β-D-mannuronic acid (M) and its C-5 epimer α-l guluronic acid (G). It consists of a mixture of GG, MG, and MM blocks, arranged in an irregular blockwise pattern that determines their properties →4 linked.2+, Sr2+ and Ba2+) of alginic acid are again insoluble in water. These properties are employed to isolate alginic acid from algae. The solubility of alginic acid in water depends on the pH of the solution, the ionic strength of the medium, and the presence of ions conditioning gelation in the solvent. As has been mentioned, alginates form acid gel and chelates with divalent cations that act as crosslinkers between adjacent polymer chains. The presence of G blocks is responsible for ionic interactions, while MG blocks are involved in the formation of weak bonds [2+ ion is most often used to prepare alginate gel. Due to their unique physicochemical properties, alginic acids and their salts have found a wide range of applications in pharmaceuticals [Alginic acid is insoluble in water but forms water-soluble salts with monovalent cations, while divalent metal salts carbodiimide (EDC)/N-hydroxysuccinimide (NHS) is particularly widely used). The average coupling efficiency ranges between 0.1 and 1.0 mol% of peptide per uronate monomer . Althougacterium . This en2+ ions.The use of alginate materials in vivo requires that, in addition to the necessary biological activity, their antibacterial properties are maintained. Current restrictions on the use of antibiotics, associated with increases in bacterial resistance, have contributed to the increasing interest in materials containing metal compounds with confirmed antibacterial properties ,36. SilvThe purpose of the current work was to obtain materials derived from copper alginate modified with RGD peptides and to assess their potential for use in the regeneration of damaged skin. It was expected that the materials would have antibacterial activity, resulting from the presence of Cu (II) compounds, but would not be cytotoxic to mammalian cells. The presence of RGD peptides was intended to improve the relevant biological properties of the materials for potential applications in regenerative medicine.2 solution, with the addition of a small amount of hydrochloric acid (0.03% HCl) at a temperature of 40 °C. A + 90% filler extract was used. Fiber stretching was performed in two stages. The first stage of the stretching process was completed in a plasticizing bath, identical in composition to the solidifying bath, at a temperature of 67 °C. The second stage of the stretching process was carried out in a superheated steam environment at a temperature of 135–140 °C. After the stretching and rinsing process, the fibers were dried at 25 °C under isometric conditions , and wetting them with distilled water and NaCl (10:1). After thorough mixing, the fibers were transferred to a Büchner funnel. After draining, round nonwoven flakes were obtained with a diameter of 4 cm .2 were obtained using Fmoc protective group protocols . Fmoc-protected amino acids were purchased from Novabiochem  or Bachem AG . As a coupling reagent, 4--4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO−) was used [2O (B) and 0.1% TFA in CH3CN (A). The HPLC spectra of crude H-RGD-OH and H-RGD-NH2 are presented in the 2 are shown in the Peptide synthesis was performed under classic solid phase conditions using 2-chlorotrityl resin and Rink-amide resin. Peptides H-RGD-OH and H-RGD-NHwas used . The pur2 peptides at concentrations of 1 mg/mL and 4 mg/mL were used for physical surface modification (spraying method) of the Cu-Alginate. The solutions (10 mL) were prepared by dissolving 10 and/or 40 mg lyophilized H-RGD-OH and/or H-RGD-NH2 respectively in Milli-Q water . Solutions of the dissolved peptides were applied by direct spraying to the copper alginate nonwovens (flakes with a diameter of 4 cm). Complete wetting of the nonwovens was determined by observing the first drops of the solution dripping from the nonwovens placed on the Buchner funnel. The modified nonwovens were dried to a constant weight in a laminar flow chamber at room temperature. In this way, copper alginate nonwovens with physically embedded peptides were obtained: H-RGD-OH  and H-RGD-NH2 . The ratios of the copper alginate to RGD derivative were 1:1 and 1:4, respectively.Solutions of H-RGD-OH and H-RGD-NH− , and N-methyl morpholine  were dissolved in dimethylformamide (DMF) (15 mL). Activation was carried out for 15 min at room temperature. The nonwoven copper alginate (0.4130 g) was treated with the solution of activated RGD. Coupling was carried out for 24 h on a shaker . The stoichiometric ratio of the used substrates was: 1:¼:¼ mmol . After 24 h, the nonwoven fabric was washed with DCM (5 mL), DMF (5 mL), DMF-water (5 mL) and CH3CN (5 mL), then dried to a constant weight. The same synthetic procedure was used to obtain Ca-Alginate-RGD, which was used in cytotoxicity assessment studies as a comparative material for Cu-Alginate-RGD.H-RGD-OH peptide , DMT/NMM/TosO−1 were obtained in the region between 400 cm−1 and 4000 cm−1. A total of 64 scans were collected, Fourier-transformed and averaged for each measurement. Recording of the spectra was performed using Bruker OPUS software (Version 6.5).FTIR ATR Spectroscopy: Infra-red spectra were obtained using a VERTEX 70 FTIR Spectrometer  with an ATR Golden Gate Diamond Accessory . Absorption spectra at a resolution of one data point per 2 cm−1. The spectra were accumulated over a 10 s integration time. Laser power was dependent on the sample and varied from 1% to 10% of power. The spectra were recorded using Renishaw WIRE 3.2 software (Version 3.2).Raman spectroscopy: Raman spectroscopy was carried out in the closed microscope chamber of the spectroscope, with the samples placed on a microscope plate. The samples were positioned in the laser light focus by means of a microscope (magnification 50×) with a charge-coupled device (CCD) camera. Raman spectra were obtained using a Renishaw InVia Reflex dispersive spectrometer with a Leica microscope . An excitation source of λ = 785 nm, 300 mW was applied, with a spectral resolution of 1 cmScanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM/EDS) analysis: Microscopic measurements of the surface topography of the materials were carried out on a VEGA3 TESCAN  scanning electron microscope equipped with an electron optics system, enabling observation of the surfaces of the materials at a 4–1,000,000-times magnification across an energy range of the incident electron beam from 0.2 keV to 30 keV with maximum resolutions of 3 nm in secondary electron emission mode and 3.5 nm in backscattered electron mode. Scanning electron microscope (SEM) images of the surface topography and cross-sections were taken using a high vacuum mode, 20 keV probe energy. The surface of each preparation was sprayed with a conductive substance—gold—using a vacuum sputter . For surface topography studies, magnifications of 300×, 1000×, 5000×, 10000× and 20,000× were used. Elemental analysis was performed using EDS INCA Energy X-ray energy dispersion spectrometer from Oxford Instruments .Staphylococcus aureus American Type Culture Collection (ATCC) 6538,Klebsiella pneumoniae ATCC 4352.Two types of materials were used in this study: Ca-Alginate-RGD  and Cu-Alginate-RGD . Antibacterial activity tests were performed according to the ISO 20743:2013 standard. Six control samples and six samples with an antibacterial compound were used. The test samples were placed in separate sterile containers and treated with 70% ethanol for a period of 30 min prior to the test. The further sterilization was performed by irradiation of both sides of the sample with UV-C light for 30 min. In accordance with the requirements of ISO 20743: 2013, the following bacterial strains were used:5 to 3.0 × 105 CFU/mL were prepared for each of the strains. Separate studies were conducted for each material. Test samples were inoculated with a bacterial suspension using 0.2 mL of the suspension for each sample. The suspension was applied to the sample in several places using a pipette. Immediately after inoculation, 20 mL of neutralizer was added to each of six samples of the control material and six samples of Cu-Alginate-RGD. Six samples (three control samples of Ca-Alginate-RGD and three test samples of Cu-Alginate-RGD) were seeded on tryptone soya agar (TSA) medium at dilutions of 100 to 10−4 to determine the initial number of bacteria per sample. Another six samples (three control samples of Ca-Alginate and three test samples Cu-Alginate-RGD) were incubated at 37 ± 2 °C for 24 h. After incubation, 20 mL of neutralizer was again added to the samples. The samples were seeded on TSA medium at dilutions of 100 to 10−4 to determine the final count bacteria per sample.Suspensions with densities from 1.0 × 102 bottles  using Eagle’s Minimum Essential Medium , supplemented with 10% horse serum . Cell culture conditions: 37 °C, atmosphere enriched with 5% CO2. The medium was changed every 3 days until a monolayer was formed. Cells for further testing were transferred using 5% trypsin-EDTA . The prepared materials were placed at the bottom of the culture wells . The L929 cells were seeded on well films with 1.5 104 cells/mL/well and cultured for 1, 3, and 7 days. Tissue culture polystyrene (TCPS) plates served as a positive control.Cell culture materials were sterilized by immersion in 70% ethanol for a period of 30 min. Subsequent sterilization consisted of irradiating both sides of the sample with UV-C light for a period of 30 min. Just before loading into the culture well, the material was washed with sterile phosphate-buffered saline . The mouse fibroblast cell line L929  was grown in 75 cmDetermination of viability—Presto Blue testSupernatant was pipetted from the wells containing cells cultured on samples of material as well as from the control wells, returning 270 µL of supernatant to each well. Next, 30 µL of PrestoBlue reagent was added to each well. The wells were then incubated for 120 min in a culture incubator.p > 0.05 were considered to be statistically significant.After incubation, 2 × 100 µL of supernatant from each of the samples of material, and the control was transferred to each of the 96 wells of an optical plate . The fluorescence intensity was measured using a microplate reader. Fluorescence intensity was calculated on the sample surface (culture well) with the test material. Statistical analysis was performed using one-way analysis of variance (ANOVA). Results with 2. The structures of the fibers were subjected to microscopic examination using a SEM microscope  using a large laboratory spinner and a 3% solution of CuClcroscope .The fibers were characterized by the numerous cracks, scratches, and crevices, which are typical for fibers formed by the wet solution method. However, this irregular surface is attractive from the point of view of surface modification with short peptides. At the same time, EDS analysis (the average of 5 samples) revealed 16.03% (Std. Deviation = 2.34) of the copper .2) were used in order to investigate whether, in the case of polysaccharide materials modified with RGD peptides, derivatives with an amide function at the C-terminus would be more active compared to H-RGD-OH [The copper alginate fibers were used to obtain nonwovens in the form of flakes with a diameter of 4 cm. Nonwovens were used as a substrate for the physical and chemical attachment of RGD derivatives. Comparison of two methods for attaching a biologically active peptide to a polysaccharide matrix should enable assessment of whether the functionalization method affects the biological activity of conjugates. Moreover, it may be expected that different functionalization methods could influence the stability of bonded peptides. In the case of a conjugate with physically embedded peptides, binding is caused only by weak interactions , while chemical functionalization should lead to significantly more stable connections, since the peptide is attached to the polysaccharide by a permanent covalent bond. Two RGD derivatives with a free carboxyl group (H-RGD-OH) and in the form of an amide  were applied. This enabled the preparation of copper alginate conjugates with physically embedded H-RGD-OH peptides  and H-RGD-NH2  with ratios copper alginate to RGD derivative of 1:1 and 1:4.The RGD derivatives used for physical modification of the copper alginate surfaces have a C-terminal amino acid in the form of an acid or amide: H-RGD-OH and H-RGD-NH−) [Attempts were also made to obtain copper alginate modified with the H-RGD-OH derivative, which is covalently linked to the polysaccharide matrix. The H-RGD-OH peptide was attached to the polysaccharide using 4--4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO−)  as a cou−) ,48, and −) ,52,53,542+ cations by monovalent ions . Moreover, the peptide H-RGD-OH was activated with DMT/NMM/TosO−, which resulted in the formation of superactive triazine esters. Thin-layer chromatography and 4-(4-nitrobenzyl)pyridine (NBP) [−. Two methods were used to assess the efficiency of conjugation of the copper alginate with the RGD derivatives: FTIR and Raman spectroscopy.The preferential formation of ester bonds between the hydroxyl groups of copper alginate and the carboxyl groups of H-RGD-OH results from the fact that synthesis proceeds under anhydrous conditions, which prevent the displacement of Cune (NBP)  were use−1, which is typical for stretching vibrations of OH groups blocks M and G . The band in the 1050 cm−1 range with the highest intensity corresponds to the skeletal vibrations of the M and G pyran rings and is characteristic of the polysaccharide glycosidic moiety.The FTIR studies began with an analysis of unmodified copper alginate. A characteristic wide band was found in the vibration range of 3500–3100 cm M and G . In addi−1 and 1550 cm−1 bands characteristic for peptide bonds (−1 characteristic for peptide (protein) compounds. Unfortunately, other characteristic bands for peptides/proteins in the 3500–3200 cm−1 range are poorly visible, due to the presence of intense bands typical for saccharides in this range .Analysis of the spectra of the copper alginate conjugates modified with RGD peptides  confirms the presence of 1650 cmde bonds . Additio2 and AlgCu-OH) obtained by physical modification, changes in the course and intensity of the bands are visible in the range of 1180–1020 cm−1. This is the range of typical polysaccharide vibrations (C-O-C glycoside bond and sugar ring)  , which i−1, which is characteristic for the amide bond (amide II) derived from RGD derivatives. In the case of the Cu-Alg-RGD conjugate which resulted from chemical modification, the range of changes is similar and relates to vibrations associated with the sugar substrate and the presence of amide bands. The intensity of the bands in the regions 1718–1584 cm−1 (amide I) and 1484–1583 cm−1 (amide II) confirms the chemical binding of RGD peptide to the alginate substrate  the maximum difference for the three controls tested immediately after inoculation was ≤1; (c) for the control sample, the value of the increase F was ≥1. Compliance with the required conditions allowed determination of the A value for Cu-Alg-RGD  compared to that of calcium alginate covalently modified with RGD (Ca-Alg-RGD), which was used as a reference. According to the criteria specified in the ISO 20743: 2013 standard, a material has significant antibacterial activity when the value for antibacterial activity A is in the range 2 ≤ A <3. When A > 3, the material is classified as highly antibacterial. The following required ISO 20743: 2013 standards were met in the tests performed using Cu-Alg-RGD as anti-bacterial material: (a) the inoculum concentration ranged from 1 × 10-Alg-RGD .S. aureus, which indicates significant antibacterial activity. For K. pneumoniae, A = 4.56, which indicates strong antibacterial activity of the product.The results of Cu-Alginate-RGD antibacterial activity were assessed at A = 2.90 for p > 0.05 were considered to be statistically significant.The L929 mouse fibroblast cell line was used in cytotoxicity assessments. Studies were conducted over seven days, with viability assessed after 1, 3 and 7 days. Presto Blue test was used, allowing assessment of the viability and proliferation of a wide range of cell types . PrestoB2 and the material with a chemically bound RGD peptide. In the case of materials obtained as a result of physical deposition of RGD derivatives, the amount of the peptide component had no significant effect on cell survival  nonwovens with the RGD derivative covalently bound to the polysaccharide matrix via an ester bond. Triazine coupling reagent was used to covalently attach the H-RGD-OH to the copper alginate. The effectiveness of both modification procedures was confirmed by spectroscopic methods (FTIR and Raman).In this study, copper alginate fibers were formed using the standard wet method, with distilled water as a solvent. The fibers contained 16.03% copper based on constant weight. The alginate fibers were used to obtain copper alginate nonwovens. The nonwovens were used as solid matrices into which were introduced RGD derivatives. Arginine-glycine-aspartate derivatives can positively affect the entire spectrum of processes in cells, from the stimulation of cell adhesion to cell migration, proliferation, survival, and apoptosis. Copper alginate conjugates with RGD derivatives were obtained by deep spraying and covalent attachment of the peptide to the polysaccharide matrix. Two types of material were obtained: (a) nonwovens with RGD derivatives H-RGD-OH and H-RGD-NHStaphylococcus aureus ATCC 6538 and Klebsiella pneumonie ATCC4352 showed that Cu-Alginate-RGD  has antibacterial activity, assessed at A = 2.90 for S. aureus, which indicates significant antibacterial activity. For K. pneumoniae, A = 4.56, which indicates strong antibacterial activity.Studies using Cytotoxicity studies on the L929 mouse fibroblast cell line using the Presto Blue test showed that the copper alginate conjugates with RGD derivatives are not cytotoxic. During incubation, an increase in cell survival was observed, which indicates that there were no adverse effects due to the presence of copper ions. Given that they also meet the requirements of biocompatibility and antibacterial activity , materials based on copper alginate conjugates with biologically active peptides show great potential for use in regenerative medicine.


DNA damage response inhibitors (DDRi) may selectively enhance the inactivation of tumor cells in combination with ionizing radiation (IR). The induction of senescence may be the key mechanism of tumor cell inactivation in this combinatorial treatment. In the current study the effect of combined IR with DDRi on the induction of senescence was studied in head and neck squamous cell carcinoma (HNSCC) cells with different human papilloma virus (HPV) status. The integrity of homologous recombination (HR) was assessed in two HPV positive, two HPV negative HNSCC, and two healthy fibroblast cell cultures. Cells were treated with the DDRi CC-115 , VE-822 , and AZD0156  combined with IR. Effects on senescence, apoptosis, necrosis, and cell cycle were analyzed by flow cytometry. The fibroblast cell lines generally tolerated IR or combined treatment better than the tumor cell lines. The ATM and ATR inhibitors were effectively inducing senescence when combined with IR. The DNA-PK inhibitor was not an important inductor of senescence. HPV status and HR activity had a limited influence on the efficacy of DDRi. Induction of senescence and necrosis varied individually among the cell lines due to molecular heterogeneity and the involvement of DNA damage response pathways in senescence induction.  Kinase inhibitors play an increasingly important role in cancer treatment . The comIn the following study, the effect of the kinase inhibitors AZD0156, inhibitor of Ataxia telangiectasia mutated kinase (ATM), VE-822, inhibitor of Ataxia telangiectasia and Rad3-related kinase (ATR) and CC-115, inhibitor of both dual mammalian target of rapamycin (mTor) and DNA-dependent protein kinase (DNA-PK), was assessed. The effect of the inhibitors in combination with IR on HPV positive and negative HNSCC tumor cells was compared to healthy tissue fibroblasts. The study focuses on cell inactivation by senescence and possible interaction effects of combined IR and DDRi.2 in a humidified atmosphere.Cal33, HSC4, UM-SCC-47, and UD-SCC-2 are HNSCC cells and were kindly provided by Dr. Thorsten Rieckmann . As described before ,9,10, CaCells were seeded on cover slides and cultured to 90% confluence maximum. Culture medium was exchanged, and half of the samples were treated with 5 µmol/L of CC-115 DNA-PK and mTor inhibitor. After 24 h of incubation at 37 °C, cells were irradiated with 10 Gy by an ISOVOLT Titan X-ray generator . After another 4 h, slides were fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100/PBS for 15 min. Slides were then blocked with 1% BSA overnight. Staining with primary antibodies mouse anti-γH2AX  and rabbit anti-Rad51  was carried out overnight at 8 °C [Cells were seeded in T25 flasks (day 0). Numbers of sewed cells were adapted to the proliferation rates of the cell lines . After 2Half of the cell culture flasks were irradiated by 2 Gy ionizing radiation (IR) 3 h after adding the drug on day 1. Change of cell culture medium and treatment with the respective inhibitors and concentrations was repeated on day 4. Cells were analyzed by flow cytometry on day 10. Streptomyces griceus , and incubated at 37 °C and 5% CO2. Bafilomycin A1 in this assay is used to inhibit the activity of endogenous beta-galactosidase by neutralizing the acidic pH of the lysosomes [12FDG by senescent-associated beta-galactosidase  showing activity at the provided unideal pH [12FDG . Incubation was continued for another hour and then samples were centrifuged , the supernatant was removed and the pellet resuspended with 200 µL of ice cold Ringer’s solution . Then, 10 µL of a 1:1 mixture of APC Annexin and 7AAD  was added and the cells incubated light-protected on ice for 30 min. Afterwards, cells were centrifuged again , resuspended in Ringer’s solution after removing the supernatant and analyzed by a CytoFLEX S flow cytometer  using PB450, FITC, PerCP, and APC channels. Data evaluation was performed using Kaluza Analysis software .Consequently, cells including the supernatant were harvested. After centrifugation  and removing of the supernatant, cells were resuspended in 400 µL of cell culture medium (2% FBS), treated with 4 µL of Bafilomycin A1 (BAF 1A), ysosomes . This alideal pH . Cells w12FDG treated cells. Senescent cells were detected among live cells only. The gate for C12FDG fluorescence was set up based on the appearance of a second C12FDG positive peak in treated HNSCC cells. Cells both positive for 7AAD and C12FDG were analyzed within the necrotic and late apoptotic cells, as we suggest they might have been in a senescent state before dying. Cells in G2/M phase of the cell cycle were identified by Hoechst 33342 . In short, the washed cells were fixed, washed again, and the cells were incubated overnight in the staining solution at 37 °C. Images were acquired by an inverse Leica DMILLED  microscope. To assess the expression of the p21 and α tubulin proteins an immune staining was performed. The cells were washed and the primary rabbit monoclonal antibodies p21  and mouse α tubulin  were incubated overnight at 4 °C. Coverslips were washed and secondary green fluorescence anti-rabbit antibodies Alexa488 and red anti-mouse antibodies Alexa594  were incubated at 37 °C for 2 h. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield . The images were acquired with the Zeiss Axio Imager Z2 fluorescence microscope at 400× magnification . Overlays were created using image processing software .Cells were cultured in T25 flasks for 10 days. Cells were then harvested, and 500 cells were taken from every flask and seeded in 60 mm dishes. Simultaneously, 500 cells were taken from a regularly split culture and seeded in 60 mm dishes. Cell culture medium was replaced after 7 days. After 14 days, cells were fixed and stained with trypan blue. Colonies consisting of at least 50 cells were counted.p-values for interaction effects detected by ANOVA are stated in the text. In the graphs, p-values of Holm–Sidak test are plotted to disaggregate ANOVA results. Graphs were generated using scientific software GraphPad Prism . Statistical comparison was performed using GraphPad Prism and SPSS . The data of clonogenic assays was analyzed by the unpaired, one-tailed Mann–Whitney U-test. Statistical analysis of the data of flow cytometry was performed by the two-way analysis of variance (two-way ANOVA) to assess the effect of IR, DDRi, and interaction effects. In the text, the term “main effect” refers to the direct effect of the independent variables single IR or single DDRi upon the dependent variables senescence, cell death, and G2 arrest, averaged across the levels of the respective other independent variable. The term “interaction effect” is used for description of combined effect of IR and DDRi on the dependent variables. Additionally to the two-way ANOVA, Holm–Sidak post-hoc multiple comparison test was applied. Homogeneity of error variances was assessed by Brown–Forsythe test. In single cases, where homogeneity of error variances was violated, we indicate it in the text. In these cases, we report only the results of the multiple comparisons and do not interpret the results of the ANOVA. p-values < 0.05 were considered statistically significant.All presented data within this paper are the mean of at least three independent experiments and shown as mean and standard deviation of the mean, unless indicated otherwise. 12FDG for senescence detection, with Hoechst 33342 for cell cycle analysis and with Annexin APC and 7AAD for apoptosis and necrosis detection  was studied by an immunostaining-based assay. γH2AX and Rad51 foci were evaluated after cells were treated with CC-115 DNA-PK inhibitor and IR. This treatment aimed to induce DNA DSBs while forcing HR-proficient cells to use HR for their repair. γH2AX marks the appearance of DSBs A. Rad51 IR alone induced senescence in healthy fibroblasts cell line SBLF9, but not SBLF7. Treatment with CC-115 and AZD0156 decreased senescence levels compared to the control as well as radiation-induced senescence in both fibroblast cell lines. There was no such effect for VE-822 A–C. As hp = 0.013). In HPV+ UD-SCC-2, there was a main effect of IR and DDRi CC-115 and AZD0156. In contrast to UD-SCC-2, both IR and DDRi decreased senescence in UD-SCC-2 (p < 0.0001), leading to a pronounced increase of senescence induction  and 0.05 µmol/L of VE-822 (p = 0.003). Necrosis was strongly increased upon combined treatment. For HPV+ UD-SCC-2, the assumption of homogeneity of variances was not met. We therefore report Holm–Sidak post hoc test results only. Both single IR and single CC-115 and AZD0156 increased necrosis in UD-SCC-2, but combined IR and DDRi did not lead to increased effect   G–I.p < 0.0001). Interestingly, a very pronounced G2 arrest was found after 2 Gy IR, persisting even after the 11 days of the assay. DDRi alone did not affect G2 arrest. There was an interaction effect for IR and VE-822 in both SBLF7 (p = 0.0108) and SBLF9 (p = 0.0018). In the fibroblast cell lines, radiation-induced G2 arrest was abrogated when IR was combined with VE-822 . Nevertheless, here the homogeneity of variances was violated and these results should be interpreted with caution. In general, the G2 arrest in HPV+ HNSCC was more pronounced in the control compared to the HPV- HNSCC , both leading to increased G2 arrest. Nevertheless, the effects of the DDRi in the HPV- HNSCC were generally small  and AZD0156 (inhibitor of ATM). Their combination with IR led to a pronounced increase of senescence in HPV+ UM-SCC-47 as well as HPV- Cal33. The induction of senescence was especially effective in Cal33, where it led to the inactivation of more than 80% of the living cells. This is particularly promising, as the prognosis of HPV- HNSCC is regarded to be less favorable as the prognosis of HPV+ HNSCC . BesidesIn contrast, IR-induced senescence was decreased upon combined treatment with AZD0156 in healthy human fibroblasts. Additionally, combined treatment did not increase necrosis in the healthy tissue fibroblasts. Combination of IR with VE-822 or AZD0156 therefore seems a promising strategy for cancer treatment. Nevertheless, these effects were observed for single cell lines and no interaction effect could be found for the other two HNSCC cell lines. As the cells were analyzed 10 days after IR in order to focus on senescence induction, we are not able to make a statement on the efficacy of combined treatment concerning apoptosis and early necrosis. A remarkable proportion of Cal33 cells was inactivated by senescence induction upon combinatorial treatment with AZD0156 and IR. Previously, HNSCC with high-risk p53 mutations have been described to assume a senescence-like phenotype and to express increased β-galactosidase staining upon combined cisplatin and Wee-1 inhibitor treatment. Wee-1 inhibition caused override of G2 arrest and sensitized p53 mutated HPV- HNSCC to cisplatin. This was resulting in mitotic arrest and senescence, but not apoptosis [A useful additional senescence marker for p53-deficient cancer cells might be the p21. p21 inhibits cyclin-dependent kinases and is involved in the establishment of cell cycle arrest in senescence . An impoIn contrast to the ATM and ATR inhibitors, we did not find an interaction effect for senescence and necrosis induction for CC-115. CC-115 is a dual mTor/DNA-PK inhibitor which has been described to effectively inhibit NHEJ via its DNA-PK inhibiting properties . DNA-PK Besides the effect on DNA-PK, the inhibition of mTor by CC-115 must also be considered. mTor signaling is involved in the regulation of numerous basic physiological processes such as metabolism, cell survival, and autophagy and is aberrantly overactivated in many cancers . mTor haParticularly important for the interpretation of senescence induction is the possible inhibitory effect of mTor on senescence via autophagy. Senescence and autophagy have been reported to be significantly associated in various cell systems . As diffRegarding cell cycle distribution, IR clearly induced a G2 arrest in the healthy fibroblast cell cultures, which persisted even after 10 days. Neither IR nor DDRi induced necrosis in the fibroblast cell lines. IR induced senescence in the SBLF9 fibroblast cell line. This may indicate that these normal tissue cells can manage DNA damage and combined treatment relatively well. We expect them to have intact cell cycle checkpoints, carrying out repair processes and inactivating unrepairable cells in a controlled manner by entering senescence. In contrast to this, in the four cancer cell lines the induction of necrosis and senescence varied largely, while there was nearly no additional G2 arrest induced by 2 Gy IR or combined treatment. This may indicate that the DNA damage processing is impaired among these cancer cells.Particularly HPV+ HNSCC have been characterized as highly radiosensitive due to inefficient DSB repair and prolonged arrest in G2 . We expeIn the present study, we found 0.05 µmol/L of VE-822 to distinctly abolish radiation-induced G2 arrest in healthy fibroblasts SBLF7 and SBLF9. There was a clear interaction effect of IR and the ATR-inhibitor on G2 arrest in these cell lines. For UM-SCC-47, G2 arrest tended to decrease similarly as in the fibroblast cell lines for VE-822 as well as AZD0156. Two-way ANOVA detected an interaction effect on G2 arrest for AZD0156 and IR for UM-SCC-4, and a main effect of single VE-822. Importantly, it has to be stated that the assumption of homogeneity of error variances was not met for G2 arrest in UM-SCC-47, so these results have to be treated with caution. We found this diminishing effect of combinatorial treatment on G2 arrest exclusively in the fibroblast cell lines and UM-SCC-47. These are the three cell lines we found to be HR-proficient. Several studies support the idea that sensitization achieved by inhibition of ATR/Chk1 is related to impairment of HR . AccordiIn our study, abrogation of G2 arrest after ATR inhibition in HR-proficient fibroblast cell lines did not lead to an increase in their sensitivity to IR, as there was no corresponding induction of senescence or necrosis. We found induction of senescence and necrosis as a result of radiosensitization in HR-proficient UM-SCC-47, but although our data suggest this might be related to abrogation of G2 arrest, this is ambiguous due to violation of homogeneity of variances. Regarding these results and the fact that ATM and ATR have been related to both HR and NHEJ , we cannWe sought to assess clear differences upon DDRi and IR treatment between HR-proficient and HR-deficient cells. In contrast to our expectations, we did not find exclusive or major efficacy of ATR inhibition in HR-proficient cell lines, nor strictly HR-dependent sensitization to IR by abrogated G2 arrest. We can only state that the studied DDRi did not induce cell death or senescence in HR-proficient healthy fibroblasts, and that senescence levels in HR-proficient UM-SCC-47 were generally low and increased only slightly upon treatment. A reason for the absence of clear differences between HR-proficient and HR-deficient cell lines could lie in the complexity of the DDR pathways. ATM and ATR have been related to both HR and NHEJ , have ovBesides the influence of HR-proficiency, we sought to identify differences in the reaction towards DDRi among HPV- and HPV+ HNSCC. Nevertheless, we found that the effects of irradiation and inhibitor treatment on senescence and necrosis varied rather individually for each cell line than in a HPV-dependent manner.HNSCC UM-SCC-47 and UD-SCC-2 are both HPV-positive. Nevertheless, upon treatment with DDRi CC-115 and VE-822, they acted reversely. The DDRi led to senescence induction in UM-SCC-47, but decreased senescence levels strongly in UD-SCC-2. Regardless of their HPV status, each HNSCC cell line was sensitive to different DDRi. Out of the two cell lines we found to react with important increase of senescence or necrosis upon combinatorial DDRi and IR treatment, one was HPV+, the other HPV-. Our findings suggest that HNSCC cell lines act rather individually upon treatment with IR and combined inhibitor treatment. A possible explanation is that there might be more relevant modifications existing in the cells, which should be taken into account besides the HPV status.The TP53 has been found to be one of the most frequently mutated genes in HNSCC , with hiCompromising p53 and pRb by viral oncoproteins might be a feature allowing HPV+ HNSCC to avoid senescence in order to drive the process of carcinogenesis . SimilarWe expected combinatorial IR and DDRi treatment to induce senescence. IR evokes DSB , which tTo conclude, the inhibition of key factors of the DDRi might have different outcomes: on the one hand, DDRi will lead to persisting DNA damage which can lead to senescence induction ,43,44. OOur main objective was to investigate whether combined IR and DDRi will induce senescence more effectively compared to single treatment. We found interaction effects for IR and DDRi for the ATM and ATR inhibitor in single cell lines. In these cases, the combined IR and DDRi enhanced senescence and necrosis induction importantly. In contrast to the ATM and ATR inhibitor, the DNA-PK and mTor inhibitor was not a major inductor of senescence and did not interact with IR. In single cell lines, particularly the induction of senescence led to an inactivation of an important proportion of the cells. Therefore, it is important to study senescence in addition to apoptosis, necrosis and cell cycle control. The fibroblast cell lines generally tolerate IR or combined treatment better than the tumor cell lines. Varying results were found for the HNSCC, probably as a result of molecular heterogeneity, complex interplay of the DDR pathways, and the involvement of DDR pathways in senescence induction. Both HPV status and HR activity appear to have a limited role in the application of DDRi. Since we found complex differences in tolerance of the combined DDRi and ionizing radiation treatment of HNSCC cell lines, further studies are needed to better understand the differences in the processing of DNA damage compared to normal tissue cells. For better comparability healthy mucosal epithelial keratinocytes should be used as controls.